Estimation of the Relative Sensitivity of qpcr Analysis Using Pooled Samples
|
|
- Conrad Manning
- 6 years ago
- Views:
Transcription
1 Estimation of the Relative Sensitivity of qpcr Analysis Using Pooled Samples Ana Muniesa 1, Chelo Ferreira 2,Héctor Fuertes 1, Nabil Halaihel 1, Ignacio de Blas 1 * 1 Department of Animal Pathology, Facultad de Veterinaria, Universidad de Zaragoza, Zaragoza, Spain, 2 Department of Applied Mathematics, Facultad de Veterinaria, Universidad de Zaragoza, Zaragoza, Spain Abstract The high sensitivity of qpcr makes it a desirable diagnostic method in epidemiological surveillance programs. However, due to high costs, the use of pooling has been suggested. In this paper, an algorithm based on the Montecarlo method has been designed and implemented. The algorithm had been tested in many different situations, and finally it was validated with a real dataset. Moreover, based on the results obtained and depending on pooling conditions, a drastic decrease of sensitivity is observed. Citation: Muniesa A, Ferreira C, Fuertes H, Halaihel N, de Blas I (2014) Estimation of the Relative Sensitivity of qpcr Analysis Using Pooled Samples. PLoS ONE 9(4): e doi: /journal.pone Editor: Joao Inacio, University of Brighton, United Kingdom Received December 17, 2013; Accepted March 5, 2014; Published April 10, 2014 Copyright: ß 2014 Muniesa et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Ministerio de Educación of Spain is funding a FPU/MEC doctorate fellowship for Ana Muniesa. Also the Departamento de Industria e Innovación del Gobierno de Aragón, Fondo Social Europeo and Dirección General de Ciencia y Tecnología (REF. MTM ) provides financial support to Research Groups A35 and T53 for their basic running costs. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * deblas@unizar.es Introduction The Polymerase Chain Reaction (PCR) is one of the most powerful technologies in molecular biology. Using PCR, specific sequences within a DNA or cdna template can be copied, or amplified, many thousand- to a million- fold. PCR is a technique requiring a specific fragment of DNA, and it is useful for different applications: to identify anomalies in the sequence of nucleotides that point to possible genetic diseases[1], to identify an individual or to determine their relationships with others or to detect the presence of DNA of microorganisms useful in the diagnosis of disease or for testing the effectiveness of a treatment [2]. In traditional (endpoint) PCR, detection of the amplified sequence are performed at the end of the reaction after the last PCR cycle, and involve post-pcr analysis such as gel electrophoresis and image analysis. In real-time quantitative PCR (qpcr), the amount of PCR product is measured at each cycle by the use of fluorescent markers that are incorporated into the PCR product [3], [2]. The increase in fluorescent signal is directly proportional to the number of PCR product molecules (amplicons) generated in the exponential phase of the reaction. Fluorescent reporters used include double-stranded DNA (dsdna)-binding dyes, or dye molecules attached to PCR primers or probes that are incorporated into the product during amplification. The change in fluorescence over the course of the reaction is measured by an equipment that combines thermal cycling with scanning capability. By plotting fluorescence against the cycle number, the qpcr equipment generates an amplification plot that represents the accumulation of product over the duration of the entire PCR reaction (Figure 1). The cycle threshold (Ct) or cycle quantification (Cq) records the cycle when the sample fluorescence exceeds a chosen threshold above background fluorescence. This value is correlated with the number of copies of the target sequence originally present in the reaction mixture [4]. The samples with a high number of initial copies of target nucleic acid, are detected sooner and therefore they will have low Ct values (usually around 20 25). However, those samples with very low numbers of copies are later detected, and the Ct values are above [5]. The sample is defined as positive when the Ct analyzed by the qpcr technique is less than the established Ct (threshold value). In other cases it is consider as negative. Using the Standard Curve Method based on known quantities, it is possible to extrapolate a value of a sample. The target DNA gene copies in the pathogen are to be considered to determine the absolute number of the agent in the processed sample, so qpcr provides us the number of copies of a particular pathogen obtained from a sample of an infected individual. The slope of the linear regression curve determines the efficiency of amplification, which is 100% if a dilution of 1:2 results in a Ct difference of 1 [6]. Currently PCR is the best-known and most successfully implemented diagnostic molecular technology. PCR, specifically qpcr, can detect slow-growing or difficult-to-culture microorganisms and can be used in situations in which clinical microbiology diagnostic procedures are inadequate, time-consuming, difficult, expensive, or hazardous to laboratory staff [2]. The analytical specificity and sensitivity of qpcr assay is considered as perfect for diagnostic of clinical cases (i.e. identification of bovine mastitis pathogens [7]). A general review over the use of qpcr in clinical microbiology testing showed an increased specificity and sensitivity over standard serological tests or culturing methods [8], and for these reasons qpcr is considered as gold standard for direct diagnosis in most of pathogens. The high sensitivity of qpcr makes it as a desirable diagnostic method to use in epidemiological surveillance programs in animal PLOS ONE 1 April 2014 Volume 9 Issue 4 e93491
2 Figure 1. Amplification plots represent the accumulation of product over the duration of the real-time PCR experiment. doi: /journal.pone g001 health [9], [10], but qpcr is a relatively expensive technique that limits its generalized application. In order to minimize this problem, the use of pooled samples has been suggested [10]. Thus, it can result in major savings (consumables and labor), and reduced costs [8], [11]. So pooling is now routinely used for health status monitoring purposes. The theoretical probability of including at least one infected individual in a pool (p infected ) is increased when the pool size (n pool ) is bigger and the prevalence (P) is higher. This probability is calculated as [12], [13]: p infected 1 ~1{(1{P) n pool However, we carefully note the significant decrease in pooled sensitivity due to the dilution effect. Two factors should be taken into account: the low proportion of infected samples in the pool (i.e. pools of big size from a population with low prevalence) and the low number of DNA copies of the infected individuals (i.e. low pathogen loads in individuals from asymptomatic populations [8]). Unfortunately, this is a common scenario of most of the epidemiological surveillance programmes: low prevalence of asymptomatic infected animals in the investigated population. In most of cases qpcr is considered as gold standard (it means that sensitivity and specifity are perfect). However, it is not completely true and the accuracy may be unknown, so it would be possible to estimate the relative sensitivity of pooled qpcr, assuming individual qpcr as gold standard. An algebraic solution of this problem is not possible and a simulation procedure is suggested. The objective of this paper is to estimate the relative sensitivity (rs) of a qpcr analysis of a pooled sample. Materials and Methods We have designed the following stochastical algorithm using the Montecarlo method. For the later convenience, we define the following variables: n pool : pool size ict: cycle threshold (defined for diagnosis of an individual sample) mct: mean of Ct in an infected and asymptomatic population. difct = ict-mct ð1þ sct: standard deviation of Ct in an infected and asymptomatic population P: prevalence of infection in an asymptomatic population Firstly, we define a pooled sample as a mix of npool individual samples. For each of the individual samples (k; k~1,...,n pool ), the infection status (infected/not infected) is randomly determinated as a function of the prevalence (P). For non-infected individual samples, the pathogen load is assumed as zero (L k ~0). For infected samples we calculate a random Ct assuming a gaussian distribution with a mean (mct) and standard deviation (sct) given Ct k ~randomðn (mct, sct) Þ: ð2þ Next, the load of an infected individual sample (L k ) is estimated by DCt-method [14]. L k ~2 (ict{ct k ) : The load of the pooled sample (L pool ) is estimated as the average of the individual samples loads (L k ) Then, the Ct pool is given as L pool ~ 1 n X pool n pool k~1 L k : Ct pool ~ log 2 L pool : As we have explained in the introduction section, when the Ct pool is lower than the ict the pool is considered as positive; in other case, as negative. During the simulation, the algorithm is iterated until a desired quantity of infected pools (I pool ) is reached. So, the number of simulations depends on the required precision for the relative sensitivity. We consider as infected pool any pool that included at ð3þ ð4þ ð5þ PLOS ONE 2 April 2014 Volume 9 Issue 4 e93491
3 Figure 2. Relative sensitivities corresponding to the simulation results for different scenarios. doi: /journal.pone g002 Figure 3. Relative sensitivities corresponding to a pooled diagnostic for PRRSv using global samples of Ct (mct = 27.75, sct = 4.625) (data from Gerber et al, 2013)[8]. doi: /journal.pone g003 PLOS ONE 3 April 2014 Volume 9 Issue 4 e93491
4 Figure 4. Relative sensitivities corresponding to a pooled diagnostic for PRRSv using low load samples of Ct (mct = 36.0, sct =1) (data from Gerber et al, 2013)[8]. doi: /journal.pone g004 least one infected individual sample. Moreover, following the previous criteria, the number of positive infected pools (P pool )is determinated. Therefore, the relative sensitivity is estimated as [12] rs~ P pool I pool The algorithm is implemented with php language (it is possible to obtain the code just asking for the authors). It has also been implemented in a web page ( php?id = 2) in order to make it available to the scientific community and biomedical practitioners. Accuracy of the results depends on the number of iterations. In order to validate the algorithm, we have used published data about prevalence of PRRS and QPCR results [8]. Results and Discussion The numerical experiments have been given for the all combinations of the next values of the variables n pool (2, 5, 10, 20), difct (2, 4, 6), sct (0.5, 1, 2), and P (1, 2, 3, 4, 5, 10, 15,, 50). The results showed in the graphics are the average of three simulations of iterations each one. By direct observation of the Figure 2, we can extract the following statements: N Both, low n pool as well as high difct, provide high sensitivity. N With high values of difct (w5), the influence of n pool is low, except for high n pool with low prevalence. N The decreasing values of difct, the increasing influence of n pool. Therefore, higher values of n pool, and low prevalence, lower values of the sensitivity. N The extreme cases are situated at the right upper zone of the Figure 2 (n pool 5 and difctƒ4). The sensitivity values are only ð6þ acceptable for very high prevalence (highly endemic diseases and epidemic outbreaks). N Finally, the lower prevalence, the higher effect of the standard deviation effect. In order to assess the consequences of pooling, we used real data from the experimental work of Gerber et al[8], about the qpcr diagnosis of PRRSv with pooled samples. Based on the individual diagnostic results from serum, the prevalences of infection, for days 1, 3, 5, 7, 14 and 21 post-infection (p.i.), were calculated. And the variabilities of Ct, in all samples globally and in a group defined as low load, were estimated. Firstly, the observed prevalences varied from 66.7% (day 1 p.i.) to 93.3{100% during the acute phase (days 3, 5 & 7 p.i.). Then, they decreased progressively to 55.6% (day 14 p.i.) and 35.6% (day 21 p.i.). When we estimated the global Ct (mct = 27.75, sct = 4.625), the high variability of the pathogen load is observed. Next, we applied our algorithm, for prevalences from 0% to 100%, to estimate the relative sensitivity in these conditions (Figure 3). In that figure, the specific values corresponding to the prevalences in 1, 3, 5, 7, 14, 21 days p.i, were marked. The relative sensitivities calculated with our method were over to 98%. Therefore, it is consistent with the results of [8]. However, these authors described a group of samples with low pathogen load (mct = 36, sct = 1). In the acute phase the relative sensitivity was greater than 90% but the marks corresponding to the 1, 14 and 21 days p.i. (early infection and recovery) where it was from 40 to 80% (Figure 4). And also this is consistent with the results of [8]. The use of pooled samples could be a good strategy in order to reduce analytical cost in surveillance programmes, but loss of sensitivity could be a critical issue due to existence of false negative results. By way of conclusion, the effect of n pool on the relative sensitivity depends on such as the values of the prevalence as the quantity of pathogen load. PLOS ONE 4 April 2014 Volume 9 Issue 4 e93491
5 Author Contributions Conceived and designed the experiments: AM CF HF NH IdB. Performed the experiments: AM CF IdB. Analyzed the data: AM CF IdB. References 1. Reiss J, Cooper DN (1990) Application of the polymerase chain reaction to the diagnosis of human genetic disease Hum Genet 85(1): Yang S, Rothman RE (2004) PCR-based diagnostics for infectious diseases: uses, limitations, and future applications in acute-care settings. Lancet Infect Dis 4(6): Abbott MA, Poiesz BJ, Byrne BC, Kwok S, Sninsky JJ, et al. (1988) Enzymatic gene amplification: qualitative and quantitative methods for detecting proviral DNA amplified in vitro. J Infect Dis 158: Bustin SA, Nolan T (2004) Pitfalls of quantitative reverse transcription polymerase chain reaction. J Biomol Tech 15: Mehta T, McGrath E, Bheemreddy S, Salimnia H, Abdel-Haq N, et al. (2010) Pranatharthi Chandrasekar and George J. Alangaden. Detection of Oseltamivir Resistance during Treatment of 2009 H1N1 Influenza Virus Infection in Immunocompromised Patients: Utility of Cycle Threshold Values of Qualitative Real-Time Reverse Transcriptase PCR. J Clin Microbiol 48(11): Carr AC, Moore SD (2012) Robust Quantification of Polymerase Chain Reactions Using Global Fitting. PLoS ONE 7(5): e Koskinen MT, Holopainen J, Pyörälä S, Bredbacka P, Pitkälä A, et al. (2009) Analytical specificity and sensitivity of a real-time polymerase chain reaction assay for identification of bovine mastitis pathogens. J Dairy Sci 92(3): Gerber PF, O Neill K, Owolodun O, Wang C, Harmon K, et al. (2013) Comparison of Commercial Real-Time Reverse Transcription-PCR Assays for Contributed reagents/materials/analysis tools: AM CF HF NH IdB. Wrote the paper: AM CF IdB. Reliable, Early, and Rapid Detection of Heterologous Strains of Porcine Reproductive and Respiratory Syndrome Virus in Experimentally Infected or Noninfected Boars by Use of Different Sample Types. J Clin Microbiol 51(2): OIE (2013) Manual of Diagnostic Tests and Vaccines for Terrestrial Animals Paris: World Organisation for Animal Health. Available: en/international-standard-setting/terrestrial-manual/access-online/. Accessed 25 February OIE (2013) Manual of Diagnostic Tests for Aquatic Animals. Paris: World Organisation for Animal Health. Available: international-standard-setting/aquatic-code/access-online/. Accessed 25 January Espy MJ, Uhl JR, Sloan LM, Buckwalter SP, Jones MF, et al. (2006) Real-Time PCR in Clinical Microbiology: Applications for Routine Laboratory Testing. Clin Microbiol Rev 19(1): Thrusfield M (2010) Veterinary Epidemiology (3rd edition). Oxford: Blackwell Science Ltd. 610 p. 13. Martin SW, Shoukri M, Thorburn MA (1992) Evaluating the health status of herds based on tests applied to individuals. Prev Vet Med 14: Schefe JH, Lehmann KE, Buschmann IR, Unger T, Funke-Kaiser H (2006) Quantitative real-time RT-PCR data analysis: current concepts and the novel gene expressions CT difference formula. J Mol Med 84(11): PLOS ONE 5 April 2014 Volume 9 Issue 4 e93491
Technical Review. Real time PCR
Technical Review Real time PCR Normal PCR: Analyze with agarose gel Normal PCR vs Real time PCR Real-time PCR, also known as quantitative PCR (qpcr) or kinetic PCR Key feature: Used to amplify and simultaneously
More informationPrincipals of Real-Time PCR. Amira A. T. AL-Hosary Lecturer of Infectious Diseases, Faculty of Veterinary Medicine, Assiut University, Egypt
Principals of Real-Time PCR Amira A. T. AL-Hosary Lecturer of Infectious Diseases, Faculty of Veterinary Medicine, Assiut University, Egypt What Is Real-Time PCR? Nucleic acid (DNA) amplification and detection
More informationqpcr Quantitative PCR or Real-time PCR Gives a measurement of PCR product at end of each cycle real time
qpcr qpcr Quantitative PCR or Real-time PCR Gives a measurement of PCR product at end of each cycle real time Differs from endpoint PCR gel on last cycle Used to determines relative amount of template
More informationQuantitative Real time PCR. Only for teaching purposes - not for reproduction or sale
Quantitative Real time PCR PCR reaction conventional versus real time PCR real time PCR principles threshold cycle C T efficiency relative quantification reference genes primers detection chemistry GLP
More informationApplications and Uses. (adapted from Roche RealTime PCR Application Manual)
What Can You Do With qpcr? Applications and Uses (adapted from Roche RealTime PCR Application Manual) What is qpcr? Real time PCR also known as quantitative PCR (qpcr) measures PCR amplification as it
More informationPolymerase chain reaction: advantages and drawbacks
Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2015 Polymerase chain reaction: advantages and drawbacks Favrot, C Posted at
More informationExecutive Summary. clinical supply services
clinical supply services case study Development and NDA-level validation of quantitative polymerase chain reaction (qpcr) procedure for detection and quantification of residual E.coli genomic DNA Executive
More informationQuantitation of mrna Using Real-Time Reverse Transcription PCR (RT-PCR)
Quantitation of mrna Using Real-Time Reverse Transcription PCR (RT-PCR) Quantitative Real-Time RT-PCR Versus RT-PCR In Real-Time RT- PCR, DNA amplification monitored at each cycle but RT-PCR measures the
More informationQPCR ASSAYS FOR MIRNA EXPRESSION PROFILING
TECH NOTE 4320 Forest Park Ave Suite 303 Saint Louis, MO 63108 +1 (314) 833-9764 mirna qpcr ASSAYS - powered by NAWGEN Our mirna qpcr Assays were developed by mirna experts at Nawgen to improve upon previously
More informationFunctional Genomics Research Stream. Research Meeting: June 19, 2012 SYBR Green qpcr, Research Update
Functional Genomics Research Stream Research Meeting: June 19, 2012 SYBR Green qpcr, Research Update Updates Alternate Lab Meeting Fridays 11:30-1:00 WEL 4.224 Welcome to attend either one Lab Log thanks
More informationHELINI Hepatitis B virus [HBV] Real-time PCR Kit (Genotype A to H)
HELINI Hepatitis B virus [HBV] Real-time PCR Kit (Genotype A to H) Quantitative In vitro diagnostics Instruction manual Cat. No: 8001-25/50/100 tests Compatible with: Agilent, Bio-Rad, Applied Bio systems
More informationPrinciples of Real Time PCR Ameer Effat M. Elfarash
Principles of Real Time PCR Ameer Effat M. Elfarash Dept. of Genetics Fac. of Agriculture, Assiut Univ. aelfarash@aun.edu.eg Types of PCR Standard PCR (conventional ) RT-PCR (Reverse Transcriptase PCR)
More informationRoche Molecular Biochemicals Technical Note No. LC 10/2000
Roche Molecular Biochemicals Technical Note No. LC 10/2000 LightCycler Overview of LightCycler Quantification Methods 1. General Introduction Introduction Content Definitions This Technical Note will introduce
More informationEfficient qpcr Setup Without Cross Contamination Using the epmotion Family of Automated Liquid Handling Systems
APPLICATION NOTE No. 368 I July 2016 Efficient qpcr Setup Without Cross Contamination Using the epmotion Family of Automated Liquid Handling Systems Eric Gancarek¹, Blandine Vanbellinghen¹, Sandrine Hamels¹,
More informationReal Time PCR (qpcr) Dott. Finetti Luca
Real Time PCR (qpcr) Dott. Finetti Luca fntlcu1@unife.it PCR (Polymerase Chain Reaction) PCR (Polymerase Chain Reaction) Theoretically: 2 n PCR (Polymerase Chain Reaction) It often used as a qualitative
More informationBIOLOGY Dr.Locke Lecture# 27 An Introduction to Polymerase Chain Reaction (PCR)
BIOLOGY 207 - Dr.Locke Lecture# 27 An Introduction to Polymerase Chain Reaction (PCR) Required readings and problems: Reading: Open Genetics, Chapter 8.1 Problems: Chapter 8 Optional Griffiths (2008) 9
More informationGuidelines for Developing Robust and Reliable PCR Assays
Guidelines for Developing Robust and Reliable PCR Assays Leta Steffen, PhD Applications Scientist Promega Corporation Outline 1) PCR reaction components What is in the reaction? How does it affect assay
More informationCurrent molecular diagnostic system
LAMP-BART Name: Chris Wong Supervisor: Prof. Margaret Ip Joint Graduate Seminar Department of Microbiology The Chinese University of Hong Kong 18 th December 2012 Current molecular diagnostic system Detection
More informationQuantitative Real time PCR. Only for teaching purposes - not for reproduction or sale
Quantitative Real time PCR PCR reaction conventional versus real time PCR real time PCR principles threshold cycle C T efficiency relative quantification reference genes primers detection chemistry GLP
More informationIntroduction to Real-Time PCR: Basic Principles and Chemistries
Introduction to Real-Time PCR: Basic Principles and Chemistries Leta Steffen, PhD Applications Scientist Promega Corporation Outline I. Real-Time PCR overview Basics of Real-Time PCR Understanding the
More informationQuantitative Real Time PCR USING SYBR GREEN
Quantitative Real Time PCR USING SYBR GREEN SYBR Green SYBR Green is a cyanine dye that binds to double stranded DNA. When it is bound to D.S. DNA it has a much greater fluorescence than when bound to
More informationReal-Time PCR: An Essential Guide
Real-Time PCR: An Essential Guide Publisher: Horizon Bioscience Editor: Kirstin Edwards, Julie Logan and Nick Saunders Genomics Proteomics and Bioinformatics Unit, Health Protection Agency, London Publication
More informationRoche Molecular Biochemicals Technical Note No. LC 12/2000
Roche Molecular Biochemicals Technical Note No. LC 12/2000 LightCycler Absolute Quantification with External Standards and an Internal Control 1. General Introduction Purpose of this Note Overview of Method
More informationReal-Time PCR Principles and Applications
Real-Time PCR Principles and Applications Dr Esam Ibraheem Azhar (BSc, MSc, Ph.D Molecular Medical Virology) Asst. Prof. Medical Laboratory Technology Department Objectives Real-Time PCR Principles and
More informationProcomcure Biotech. PCR Reagents
Procomcure Biotech PCR Reagents valid for 2018 VitaTaq DNA Polymerase is a standard Taq DNA polymerase suitable for all common PCR applications like colonypcr, cloning applications, high-throughput PCR
More informationReal Time PCR. Group Members: Alanna, Susan, Jane, Sam & Rachel
Real Time PCR Group Members: Alanna, Susan, Jane, Sam & Rachel General Overview of Standard PCR 1. Temperature raised to 95 C where double stranded DNA becomes single strands 2. Temperature lowered to
More informationInternal controls for real-time PCR-based pathogen identification
Internal controls for real-time PCR-based pathogen identification QIAGEN Dr. Lillian Roth Lead Scientist Veterinary R&D Team Global R&D Competence Center Applied Testing QIAGEN webinar June 1, 2011 Introduction
More informationMolecular methods for detection of Antibiotic Resistance in environmental matrices: limits, prospects and challenges.
Dr. Angela Cicatelli acicatelli@unisa.it Molecular methods for detection of Antibiotic Resistance in environmental matrices: limits, prospects and challenges. 1st Workshop on "Risk prognosis of environmental
More informationPolymerase Chain Reaction: Application and Practical Primer Probe Design qrt-pcr
Polymerase Chain Reaction: Application and Practical Primer Probe Design qrt-pcr review Enzyme based DNA amplification Thermal Polymerarase derived from a thermophylic bacterium DNA dependant DNA polymerase
More informationINTRODUCTION TO REVERSE TRANSCRIPTION PCR (RT-PCR) ABCF 2016 BecA-ILRI Hub, Nairobi 21 st September 2016 Roger Pelle Principal Scientist
INTRODUCTION TO REVERSE TRANSCRIPTION PCR (RT-PCR) ABCF 2016 BecA-ILRI Hub, Nairobi 21 st September 2016 Roger Pelle Principal Scientist Objective of PCR To provide a solution to one of the most pressing
More informationLoop-mediated Isothermal Amplification (LAMP) as a diagnostic tool in detection of infectious diseases
Loop-mediated Isothermal Amplification (LAMP) as a diagnostic tool in detection of infectious diseases Dorota DRAPAŁA, Milena KORDALEWSKA Keywords: LAMP; DNA amplification; isothermal reaction Abstract:
More informationLe linee guida MIQE. Paola Scaruffi. S. S. Oncologia Traslazionale Pediatrica Istituto Nazionale per la Ricerca sul Cancro (IST) Genova
Le linee guida MIQE Paola Scaruffi S. S. Oncologia Traslazionale Pediatrica Istituto Nazionale per la Ricerca sul Cancro (IST) Genova Technology 1997 Legacy PCR Qualitative assay Novelty free-for-all Biology
More informationDevelopment of Improved
The Essentials of Life Science Research Globally Delivered Development of Improved Synthetic Molecular Standards for Norovirus Genogroups I and II Shamaila Ashraf, Ph.D., Prasanthi Bandi, M.S., Brian Chase,
More informationSupplementary Information
Electronic Supplementary Material (ESI) for Lab on a Chip. This journal is The Royal Society of Chemistry 2016 Supplementary Information Translating Diagnostic Assays from the Laboratory to the Clinic:
More informationP HENIX. PHENIX PCR Enzyme Guide Tools For Life Science Discovery RESEARCH PRODUCTS
PHENIX PCR Enzyme Guide PHENIX offers a broad line of premium quality PCR Enzymes. This PCR Enzyme Guide will help simplify your polymerase selection process. Each DNA Polymerase has different characteristics
More informationCulture Vs. qpcr. Daniel Grandio Gonzalez
Culture Vs. qpcr Daniel Grandio Gonzalez Introduction Legionelosis, Pontiac Fever, Mycobacteriosis Legionella: Hot water tanks, showers, cooling towers, swimming pools Environmental Mycobacteria: Purified
More informationBootcamp: Molecular Biology Techniques and Interpretation
Bootcamp: Molecular Biology Techniques and Interpretation Bi8 Winter 2016 Today s outline Detecting and quantifying nucleic acids and proteins: Basic nucleic acid properties Hybridization PCR and Designing
More informationThe following is an overview of diagnostic techniques for Ebola infection in humans.
IBM Deep Dive Topic 1/14/2015 Alex // operonlabs.com Diagnostics and Ebola: The following is an overview of diagnostic techniques for Ebola infection in humans. Current Status: The current status of Ebola
More informationBeginner s guide to Real-time PCR
Beginner s guide to Real-time PCR Beginner s guide to Real-time PCR PCR or the Polymerase Chain Reaction has become the cornerstone of modern molecular biology the world over. Real-time PCR is a bespoke
More informationMethods in virus diagnosis PCR techniques
Methods in virus diagnosis PCR techniques 450 MBIO PRACTICAL LESSON 5 Molecular Methods Methods based on the detection of viral genome are also commonly known as molecular methods. It is often said that
More informationIndex 273 Index A Acrylamide gel electrophoresis Trichomonas vaginalis, 231, 234 Agarose gel electrophoresis amplification detection, 3 HCV detection,
Index 273 Index A Acrylamide gel electrophoresis Trichomonas vaginalis, 231, 234 Agarose gel electrophoresis amplification detection, 3 HCV detection, 167, 169, 170 Legionella species, 175, 177, 179, 180
More informationLATE-PCR. Linear-After-The-Exponential
LATE-PCR Linear-After-The-Exponential A Patented Invention of the Laboratory of Human Genetics and Reproductive Biology Lab. Director: Lawrence J. Wangh, Ph.D. Department of Biology, Brandeis University,
More informationQUANTITATIVE RT-PCR PROTOCOL (SYBR Green I) (Last Revised: April, 2007)
QUANTITATIVE RT-PCR PROTOCOL (SYBR Green I) (Last Revised: April, 007) Please contact Center for Plant Genomics (CPG) facility manager Hailing Jin (hljin@iastate.edu) regarding questions or corrections.
More informationFor in vitro Veterinary Diagnostics only. Kylt Brachyspira spp. Real-Time PCR Detection.
For in vitro Veterinary Diagnostics only. Kylt Brachyspira spp. Real-Time PCR Detection www.kylt.eu DIRECTION FOR USE Kylt Brachyspira spp. Real-Time PCR Detection A. General Kylt Brachyspira spp. products
More information2 march 06 Seminar on RT-PCR. About Real-time PCR. Aurélie OLIVIER Université Catholique de Louvain Unité de pharmacologie cellulaire et moléculaire
2 march 06 Seminar on RT-PCR About Real-time PCR Aurélie OLIVIER Université Catholique de Louvain Unité de pharmacologie cellulaire et moléculaire Target DNA PCR Applications: Gene Plasmide, phage Diagnostic
More informationIntroduction To Real-Time Quantitative PCR (qpcr)
Introduction To Real-Time Quantitative PCR (qpcr) Samuel Rulli, Ph.D. Samuel.Rulli@QIAGEN.com Technical Support: BRCsupport@qiagen.com The products described in this webinar are intended for molecular
More informationRoche Applied Science Technical Note No. LC 10/update 2003
Roche Applied Science Technical Note No. LC 10/update 2003 LightCycler Overview of LightCycler Quantification Methods Purpose of this Note Over the last few years, real-time-pcr has become essential for
More informationReal-Time PCR Workshop Gene Expression. Applications Absolute and Relative Quantitation
Real-Time PCR Workshop Gene Expression Applications Absolute and Relative Quantitation Absolute Quantitation Easy to understand the data, difficult to develop/qualify the standards Relative Quantitation
More informationChapter 20 Recombinant DNA Technology. Copyright 2009 Pearson Education, Inc.
Chapter 20 Recombinant DNA Technology Copyright 2009 Pearson Education, Inc. 20.1 Recombinant DNA Technology Began with Two Key Tools: Restriction Enzymes and DNA Cloning Vectors Recombinant DNA refers
More informationReal Time Quantitative PCR Assay Validation, Optimization and Troubleshooting
Real Time Quantitative PCR Assay Validation, Optimization and Troubleshooting Dr Steffen Muller Field Applications Scientist Stratagene Europe Overview The Importance of Controls Validation and Optimization
More informationQ: Using at least 3 biological replicates in an experiment is recommended to do. What do you suggest: At which step of calculation of the relative
The questions below have been asked by attendees of the qpcr webinar series, Part 2: Analyzing Your Data. All the questions, including the questions that could not be answered during the webinar have been
More informationSite directed mutagenesis, Insertional and Deletion Mutagenesis. Mitesh Shrestha
Site directed mutagenesis, Insertional and Deletion Mutagenesis Mitesh Shrestha Mutagenesis Mutagenesis (the creation or formation of a mutation) can be used as a powerful genetic tool. By inducing mutations
More informationBioengineering of Tobacco Mosaic Virus to Create a Non- Infectious Positive Control for Ebola Diagnostic Assays
Supplementary Information for Bioengineering of Tobacco Mosaic Virus to Create a Non- Infectious Positive Control for Ebola Diagnostic Assays Patricia Lam 1, Neetu M. Gulati 2,3, Phoebe L. Stewart 2,3,
More informationCHAPTER 3 DEVELOPMENT OF DENV GROUP SPECIFIC REAL TIME RT-PCR
CHAPTER 3 DEVELOPMENT OF DENV GROUP SPECIFIC REAL TIME RT-PCR 28 Dengue is diagnosed by either detecting virus or antibody to the virus in blood. Isolation of virus in cell culture or in infant mouse brain
More informationThe Polymerase Chain Reaction. Chapter 6: Background
The Polymerase Chain Reaction Chapter 6: Background PCR Amplify= Polymerase Chain Reaction (PCR) Invented in 1984 Applications Invention of PCR Kary Mullis Mile marker 46.58 in April of 1983 Pulled off
More informationIn the name of God. Application of Molecular Methods for Detection of Viral Pathogens & Viral Resistance
In the name of God Application of Molecular Methods for Detection of Viral Pathogens & Viral Resistance By: Kiana Shahzamani From :Digestive Disease Research Center (DDRC) Why detect microorganisms? Determine
More informationStandardisation of real-time PCR. Dr P. Lewis White Public Health Wales
Standardisation of real-time PCR Dr P. Lewis White Public Health Wales The MIQE Guidelines Publications reporting real-time PCR are ubiquitous Lack of sufficient experimental detail Limits the potential
More informationThe following supplement accompanies the article
The following supplement accompanies the article Recommended reporting standards for test accuracy studies of infectious diseases of finfish, amphibians, molluscs and crustaceans: the STRADAS-aquatic checklist
More informationMIqPCR: Minimum Information about a Quantitative Polymerase Chain Reaction experiment Version 1.0, 4th April, 2008.
MIqPCR: Minimum Information about a Quantitative Polymerase Chain Reaction experiment Version 1.0, 4th April, 2008. RDML-Consortium This module identifies the minimum information required to report the
More informationModule1TheBasicsofRealTimePCR Monday, March 19, 2007
Objectives Slide notes: Page 1 of 41 Module 1: The Basics Of Real Time PCR Slide notes: Module 1: The Basics of real time PCR Page 2 of 41 Polymerase Chain Reaction Slide notes: Here is a review of PCR,
More informationUltraFast Molecular Diagnostic System
UltraFast Molecular Diagnostic System CONTENTS 01 PCR vs Real-time PCR 02 NANOBIOSYS Sample Prep G2-16TU 03 NANOBIOSYS Real-time PCR G2-4 01 PCR vs Real-time PCR What is DNA & What is PCR? NANOBIOSYS 4
More informationApplication Note Detecting low copy numbers. Introduction. Methods A08-005B
Application Note Detecting low copy numbers A08-005B Introduction Sensitivity of a qpcr assay is highly dependent on primer efficiency. Not all assays will be capable of detecting a single copy of template
More informationAPPLICATION NOTE. MagMAX mirvana Total RNA Isolation Kit TaqMan Advanced mirna Assays
APPLICATION NOTE MagMAX mirvana Total RNA Isolation Kit TaqMan Advanced mirna Assays A complete workflow for high-throughput isolation of serum mirnas and downstream analysis by qrt-pcr: application for
More informationLATE-PCR and Allied Technologies for Analysis of Genetic Heterogeneity in Single Cells & Single DNA Molecules
Building Tools for the Analysis of Cellular Heterogeneity LATE-PCR and Allied Technologies for Analysis of Genetic Heterogeneity in Single Cells & Single DNA Molecules J. Aquiles Sanchez, Adam E. Osborne,
More informationReal-Time PCR Detection Reagents for Detection of Marek Disease Virus and differentiation between field strains and Rispens vaccine CVI988 strain
For in vitro Veterinary Diagnostics only. Real-Time PCR Detection Reagents for Detection of Marek Disease Virus and differentiation between field strains and Rispens vaccine CVI988 strain www.kylt.eu DIRECTION
More informationChapter 17. PCR the polymerase chain reaction and its many uses. Prepared by Woojoo Choi
Chapter 17. PCR the polymerase chain reaction and its many uses Prepared by Woojoo Choi Polymerase chain reaction 1) Polymerase chain reaction (PCR): artificial amplification of a DNA sequence by repeated
More informationUser Manual. NGS Library qpcr Quantification Kit (Illumina compatible)
NGS Library qpcr Quantification Kit (Illumina compatible) User Manual 384 Oyster Point Blvd, Suite 15 South San Francisco, CA 94080 Phone: 1 (888) MCLAB-88 Fax: 1 (650) 872-0253 www.mclab.com Contents
More informationFUTURE PROSPECTS IN MOLECULAR INFECTIOUS DISEASES DIAGNOSIS
FUTURE PROSPECTS IN MOLECULAR INFECTIOUS DISEASES DIAGNOSIS Richard L. Hodinka, Ph.D. University of South Carolina School of Medicine Greenville Greenville Health System, Greenville, SC hodinka@greenvillemed.sc.edu
More informationOnly for teaching purposes - not for reproduction or sale
PCR reaction conventional versus real time PCR real time PCR principles threshold cycle C T efficiency relative quantification reference genes primers detection chemistry GLP in real time PCR Relative
More informationFor in vitro Veterinary Diagnostics only. Kylt Avian Hepatitis E. Real-Time RT-PCR Detection.
For in vitro Veterinary Diagnostics only. Kylt Avian Hepatitis E Real-Time RT-PCR Detection www.kylt.eu DIRECTION FOR USE Kylt Avian Hepatitis E Real-Time RT-PCR Detection A. General Kylt Avian Hepatitis
More informationMolecular-level dengue fever diagnostic devices made out of paper
Supplementary Information Molecular-level dengue fever diagnostic devices made out of paper Shih-Jie Lo a, Shih-Chun Yang b, Da-Jeng Yao a, Jiann-Hwa Chen b, Wu-Chun Tu c, Chao-Min Cheng a * a Institute
More informationA peer-reviewed version of this preprint was published in PeerJ on 16 March 2018.
A peer-reviewed version of this preprint was published in PeerJ on 16 March 2018. View the peer-reviewed version (peerj.com/articles/4473), which is the preferred citable publication unless you specifically
More informationReal-time RT-PCR Test Kit for Detection of Classical Swine Fever Virus
V3_2011-03-21 VIROTYPE CSFV Instructions for Use Real-time RT-PCR Test Kit for Detection of Classical Swine Fever Virus In vitro Diagnostic Kit for Swine Registered in accordance with 17c of the German
More information13-2 Manipulating DNA Slide 1 of 32
1 of 32 The Tools of Molecular Biology The Tools of Molecular Biology How do scientists make changes to DNA? Scientists use their knowledge of the structure of DNA and its chemical properties to study
More informationThe Polymerase Chain Reaction. Chapter 6: Background
The Polymerase Chain Reaction Chapter 6: Background Invention of PCR Kary Mullis Mile marker 46.58 in April of 1983 Pulled off the road and outlined a way to conduct DNA replication in a tube Worked for
More informationMolecular Cell Biology - Problem Drill 11: Recombinant DNA
Molecular Cell Biology - Problem Drill 11: Recombinant DNA Question No. 1 of 10 1. Which of the following statements about the sources of DNA used for molecular cloning is correct? Question #1 (A) cdna
More informationPCR validation. August 2014 Willem van Leeuwen
PCR validation August 2014 Willem van Leeuwen Validation of PCR test Primers, probes has been designed. PCR protocol developed. Does the new developed assay meet all performance criteria? Is the outcome
More informationAbout the LIAISON MDX
The quality of treatment starts with diagnosis USA Molecular Product MENU Simplexa Molecular Kits LIAISON MDX Molecular Reagents Primer Pairs US. Molecular Menu About the LIAISON MDX The LIAISON MDX is
More informationPrimePCR Assay Validation Report
Gene Information Gene Name Gene Symbol Organism Gene Summary Gene Aliases RefSeq Accession No. UniGene ID Ensembl Gene ID mcf.2 transforming sequence-like Mcf2l Mouse Description Not Available C130040G20Rik,
More informationMOLECULAR TESTING: VERIFYING/VALIDATING INSTRUMENTS, REAGENTS AND ASSAYS. Richard L. Hodinka, Ph.D.
MOLECULAR TESTING: VERIFYING/VALIDATING INSTRUMENTS, REAGENTS AND ASSAYS Richard L. Hodinka, Ph.D. University of South Carolina School of Medicine Greenville Greenville Health System, Greenville, SC hodinka@greenvillemed.sc.edu
More informationBio Rad PCR Song Lyrics
Bio Rad PCR Song Lyrics There was a time when to amplify DNA, You had to grow tons and tons of tiny cells. (Oooh) Then along came a guy named Dr. Kary Mullis, Said you can amplify in vitro just as well.
More informationEVALUATION OF AUTOMATED RT-PCR SYSTEMS TO ACCELERATE FMD DIAGNOSIS
Appendix 25 EVALUATION OF AUTOMATED RT-PCR SYSTEMS TO ACCELERATE FMD DIAGNOSIS Scott M. Reid, Nigel P. Ferris, Geoffrey H. Hutchings and Soren Alexandersen Institute for Animal Health, Pirbright Laboratory,
More informationAromatase Activity and CYP19 Gene Expression in male adult Xenopus laevis Exposed to Atrazine
Aromatase Activity and CYP19 Gene Expression in male adult Xenopus laevis Exposed to Atrazine June-Woo Park*, Markus Hecker*, Margaret Murphy*, Paul Jones*, Glen van der Kraak**, Keith Solomon**, Ron Kendall***,
More informationModeling DNA Amplification Technology for Process Optimization.
Modeling DNA Amplification Technology for Process Optimization. Mentor: Dr. Emily Stone, University of Montana. Group Neil Olver, McGill University Jon Collis, RPI Yevgeniy Frenkel, RPI Derek Moulton,
More informationDetection and Quantitation of Residual Host Cell DNA
Detection and Quantitation of Residual Host Cell DNA White Paper Author: Phil Kuhlman, Biopharmaceutical Technical Specialist 1 Abstract All biological drug products are required to be characterised for
More informationFor in vitro Veterinary Diagnostics only. Kylt Avian Nephritis Virus. Real-Time RT-PCR Detection.
For in vitro Veterinary Diagnostics only. Kylt Avian Nephritis Virus Real-Time RT-PCR Detection www.kylt.eu DIRECTION FOR USE Kylt Avian Nephritis Virus Real-Time RT-PCR Detection A. General Kylt Avian
More informationi) Wild animals: Those animals that do not live under human supervision or control and do not have their phenotype selected by humans.
The OIE Validation Recommendations provide detailed information and examples in support of the OIE Validation Standard that is published as Chapter 1.1.6 of the Terrestrial Manual, or Chapter 1.1.2 of
More informationKayhan Azadmanesh MD, Ph.D. Virology Department
Principles of molecular tests Kayhan Azadmanesh MD, Ph.D. Virology Department Pasteur Institute t of Iran 1 DNA molecule (1954) 2 Southern Blot (1975) 3 We needed to know the sequence of the genes and
More informationGeneCopoeia TM. All-in-One qpcr Mix For universal quantitative real-time PCR. User Manual
GeneCopoeia TM Expressway to Discovery All-in-One qpcr Mix For universal quantitative real-time PCR Cat. No. AOPR-0200 (200 qpcr reactions) Cat. No. AOPR-0600 (600 qpcr reactions) Cat. No. AOPR-1000 (1000
More informationFor in vitro Veterinary Diagnostics only. Real-Time RT-PCR Detection Kit for beta-actin.
For in vitro Veterinary Diagnostics only. Real-Time RT-PCR Detection Kit for beta-actin www.kylt.eu DIRECTION FOR USE Art. No. 31106 / 31107 Kylt Host Cells Real-Time RT-PCR Detection Kit for beta-actin
More informationGoing MULTI How to Easily Achieve High Multiplexing in Real-Time PCR
qpcr Symposium 2005 - Weihenstephan Going MULTI How to Easily Achieve High Multiplexing in Real-Time PCR Dr. Andreas Missel Associate Director Research & Development Overview Introduction Multiplex real-time
More informationFor in vitro Veterinary Diagnostics only. Kylt Influenza A - H9. Real-Time RT-PCR Detection.
For in vitro Veterinary Diagnostics only. Kylt Influenza A - H9 Real-Time RT-PCR Detection www.kylt.eu DIRECTION FOR USE Kylt Influenza A - H9 Real-Time RT-PCR Detection A. General Kylt Influenza A - H9
More informationResearch Article Extraction of HCV-RNA from Plasma Samples: Development towards Semiautomation
International Analytical Chemistry Volume 2015, Article ID 367801, 4 pages http://dx.doi.org/10.1155/2015/367801 Research Article Extraction of HCV-RNA from Plasma Samples: Development towards Semiautomation
More informationHiPer RT-PCR Teaching Kit
HiPer RT-PCR Teaching Kit Product Code: HTBM024 Number of experiments that can be performed: 5 Duration of Experiment: Protocol: 4 hours Agarose Gel Electrophoresis: 45 minutes Storage Instructions: The
More informationAmoyDx TM RRM1 Gene Expression Analysis Kit
AmoyDx TM RRM1 Gene Expression Analysis Kit Instructions For Use Instructions Version: B2.1 Date of Revision: April 2012 Store at -20±2 o C -1/5- Background RRM1 is one of the targets of Gemcitabine, and
More informationFLUXERGY ANALYZER BETA A MODULAR PLATFORM FOR POINT-OF-CARE PCR
ß BETA FLUXERGY ANALYZER BETA A MODULAR PLATFORM FOR POINT-OF-CARE PCR Fluxergy LLC 13766 Alton Parkway Suite 15 Irvine, CA 92618 USA (949) 5-41 inquire@fluxergy.com www.fluxergy.com OBJECTIVE To introduce
More informationqpcr course Alyona Minina Department of Plant Biology SLU
qpcr course Alyona Minina Department of Plant Biology SLU 2014 What do you want to know? Data Analysis and interpretation Statistical analysis of qpcr How qpcr works Application of qpcr Basics Normalization
More informationApplied Biosystems Real-Time PCR Rapid Assay Development Guidelines
Applied Biosystems Real-Time PCR Rapid Assay Development Guidelines Description This tutorial will discuss recommended guidelines for designing and running real-time PCR quantification and SNP Genotyping
More informationPrimePCR Assay Validation Report
Gene Information Gene Name collagen, type IV, alpha 1 Gene Symbol Organism Gene Summary Gene Aliases RefSeq Accession No. UniGene ID Ensembl Gene ID COL4A1 Human This gene encodes the major type IV alpha
More informationESCMID Online Lecture Library. by author
Molecular diagnostics: when to use (commercial vs in house) Marijke Raymaekers (Belgium) Kate Templeton (UK) Are you mainly using home-brew or commercial assays? In house diagnostics home brew assays Commercial
More information